We have previously identified sets of tissue-specific microRNAs that regulate metastatic progression by distinct cancer types. In both breast cancer and melanoma, such metastasis regulating miRNA pathways enable cancer cells to avidly recruit endothelial cells into the primary tumor site (Png et al., Nature, 2011; Pencheva et al., Cell, 2012). A key unknown in the field has been the signal(s) that are provided by such recruited endothelial cells that impact metastatic progression. We have used an innovative unbiased approach wherein ribosomes of tumor endothelial cells are genetically marked with an affinity tag. This enables purification of tumor endothelial ribosomes along with their associated transcripts, which then undergo next-generation sequencing. This allowed us to identify Slit2 as a gene significantly induced in endothelial cells by highly metastatic cells. Slit2 is an axon guidance molecule required for the proper establishment of nervous system connectivity. Our preliminary evidence in syngeneic models reveals that genetic inactivation of Slit2 in the endothelial compartment significantly impairs cancer metastasis from the primary tumor site. We propose a model whereby metastatic cells induce Slit2 in endothelial cells, which serves as a signal that promotes migration of cancer cells within the tumor (low Slit2) towards the vasculature (high Slit2), enabling intravasation and metastasis. This model is supported by preliminary clinical association evidence that reveals that increased Slit2 in endothelial compartment relative to the tumoral compartment associates with higher stage tumors that exhibit higher rates of metastatic relapse. In this application, we propose a series of complementary approaches for rigorously confirming this surprising model and further mechanistically dissecting it. We will modulate Slit2 signal sensing by cancer cells through genetic inactivation of endothelial or tumoral Slit2 using cell-type specific genetic inactivation in a genetically initiated model of cancer progression. We will employ live animal multi-photon microscopy to visualize Slit2-driven tumoral trans-endothelial migration and intravasation. We aim to identify the tumoral receptor that senses Slit2, to use immunohistochemical methods to investigate an association between endothelial Slit2 and human cancer progression and metastatic relapse, and to discover the tumor-derived signal that induces endothelial Slit2. Finally, we will apply these insights by determining if a clinically used therapeutic, which we find induces Slit2 promotes cancer metastasis. This work has the potential for major impact on our understanding of mechanisms of cancer progression by establishing endothelial cells as major orchestrators of metastasis. It could have important impact on human disease given that this pathway governs progression of highly prevalent cancer types and associates with human relapse. Moreover, the cell-type specific ribosomal profiling method we have employed could be applied more broadly to study endless cell-types within the tumor microenvironment.